1. Field of the Invention
This invention pertains to a biosensor for detecting and/or quantifying analytes. More particularly, this invention pertains to a universal biosensor based on activity-dependent changes in enzyme electrostatic fields and the methods of their detection.
2. Description of Related Art
Biosensors are devices that can detect and/or quantify analytes using known interactions between a targeted analyte and a biological macromoleculer binding agent such as an enzyme, receptor, DNA or antibody. Biosensors are unique in that they have applications in virtually all areas of human endeavor. In example, biosensors have utility in fields as diverse as blood glucose monitoring for diabetics, the recognition of poisonous gas and/or explosives, the detection of chemicals commonly associated with spoiled or contaminated food, genetic screening, and environmental testing.
Biosensors are commonly categorized according to two features, namely, the type of macromolecule utilized in the device and the means for detecting the moment contact occurs between the binding agent and the targeted analyte. Major classes of biosensors include enzyme (or catalytic) biosensors, immunosensors and DNA biosensors.
Enzyme (or catalytic) biosensors utilize one or more enzymes as the macromolecule and take advantage of the complimentary shape of the selected enzyme and the targeted analyte. Enzymes are proteins that perform most of the catalytic work in biological systems and are known for highly specific catalysis. The shape and reactivity of a given enzyme limit its catalytic activity to a very small number of possible substrates. Enzymes are also known for speed, working at rates as high as 10,000 conversions per second per enzyme molecule. Enzyme biosensors rely on the specific chemical changes related to the enzyme/analyte interaction as the means for recognizing contact with the targeted analyte. For example, upon interaction with an analyte, a biosensor may generate electrons, a colored chromophore or a change in pH as the result of the relevant enzymatic reaction. Alternatively, upon interaction with an analyte, an enzyme may cause a change in a fluorescent or chemiluminescent signal that can be recorded by an appropriate detection system.
Immunosensors utilize binding agents called antibodies. Antibodies are protein molecules that generally do not perform catalytic reactions but are designed to bind with specific foreign molecules, called antigens, that are at times associated with some disease states. Antibodies attach to antigens and either remove the antigens from the host and/or trigger an immune response. Antibodies are quite specific in their interactions and, unlike enzymes, they are capable of recognizing and selectively binding to very large bodies such as single cells. Thus, antibody-based biosensors allow for the identification of certain pathogens such as dangerous bacterial strains.
DNA biosensors utilize the complimentary nature of the DNA or RNA double-strands and are designed for the detection of DNA or RNA fragments usually associated with a given medical condition. A sensor generally uses a single-strand from a DNA double helix as the binding agent. The nucleic acid material in a given test sample is then denatured and placed into contact with the binding agent. If the strands in the test sample are complementary to the strands used as the binding agent, the two interact. The interaction can be monitored by various means such as a change in mass at the sensor surface or the presence of a fluorescent or radioactive signal. Alternative arrangements have binding of the sample of interest to the sensor and subsequent treatment with labeled probes to allow for identification of the sequence(s) of interest.
While the potential utility for biosensors is great and while hundreds of biosensors have been described in patents and in the literature, actual commercial use of biosensors remains limited. All enzyme biosensor designs set forth in the prior art contain one or more inherent weaknesses. Some lack the sensitivity and/or speed of detection necessary to accomplish certain tasks. Other such biosensors lack long-term stability. Still other biosensors cannot be sufficiently minaturized to be commercially viable or are prohibitively expensive to produce. Often biosensors must be pre-treated with salts and/or enzyme cofactors, a practice that is inefficient and bothersome. To date, virtually all enzyme biosensors are limited by the chemistries they employ to monitor contact between the sensing enzyme and the targeted analytes, regardless of whether the chemistry causes a pH change (generation of protons), a color change (generation or removal or a colored chromophore) or a change in oxidation state. One system takes advantage of a fluorescence change that occurs when glucose binds to a fluorescently-tagged glucose-binding protein. This system is hampered in its requirement for structural data and site-directed mutagenesis for the proper placement of the fluorescent probe.
The following references represent the closest prior art to the instantly claimed invention known to applicant as of the filing date of the instant application. We note that the binding of enzymes to solid supports is not new art; publications on the subject can be found in books and articles for well over two decades. Methods for enzyme immobilization for use in biosensor device construction have also been frequently reported in the literature and patents. The novel aspect of the instant invention is the chemistry-independent nature of the detection of enzyme action and thus substrate (analyte) presence:
(1) U.S. Pat. No. 5,156,810 to Ribi and U.S. Pat. No. 5,491,097 to Ribi et al. (hereinafter referenced as "the Ribi patents"). The Ribi patents teach biosensors employing a thin crystalline diyne surfactant polymeric electrically conducting layer. Specific binding pairs may be bound to the layer. Binding of an analyte or a reagent to the specific binding pair member layer may change the electrical, optical or structural properties of the layer for measurement of the analyte. The change in the polymeric layer provides for a measurement.
Contrary to the instant invention, the Ribi patents do not detect analytes by monitoring changes in voltages arising from deviations in the electrostic field surrounding an enzyme as it moves during contact with its substrate(s). In fact, the Ribi patents do not even indicate a preference for using an enzyme as the binding pair member. In the Ribi patents, the diyne binding layer is the critical detection component, while in the instant invention detection occurs away from the enzyme and its chemical supports. Any change in electrical property and/or conductivity referenced in the Ribi patents is due to charges that are released during specific chemical interactions between a binding pair rather than voltage or electromagentic radiation changes due to enzyme movements in response to the presence of the molecule of interest.
(2) Immobilization of Protein Molecules onto Homogeneous and Mixed Carboxylate-Terminated Self-Assembled Monolayers by Patel et al., Langmuir 1997, 6485-6490 (hereinafter referenced as "Patel"). Patel teaches binding of the protein catalase to gold surfaces modified by self-assembled monolayers (SAMs). Patel also teaches that the attachment of biomolecules, in particular proteins, onto solid supports is fundamental in the development of advanced biosensors. However, Patel does not teach an enzyme biosensor that measures contact with a targeted analyte by monitoring changes in voltage arising from deviations in the electrostatic field around enzymes as they move during contact with an analyte. In fact, Patel does not teach a functional biosensor of any sort, although as in all biosensors, detection of enzyme function depends on the chemistry performed by the bound enzyme, in this case, catylase. PA1 (3) Wilner and his co-workers (Assembly of Functionalized Monolayers of Redox Proteins on Electrode Surfaces: Novel Bioelectronic and Optobioelectronic Systems, Biosensors & Bioelectronics 1997, 337-356; NAD.sup.+ -Dependent Enzyme Electrodes: Electrical Contact of Cofactor- Dependent Enzymes and Electrodes, J. Am. Chem. Soc., 1997, 9114-9119) have used SAM's to anchor enzymes to gold electrodes. Yet, their biosensors rely on electron transfer from the sensing enzyme directly or through the SAM intermediate to the gold electrode. All sensors that employ an enzyme "oxidase" (an enzyme that performs chemistry that involves electron generation and/or transfer) alone or in tandem with other enzyme species will rely on oxidation-reduction chemistries by the oxidase to detect the molecule (glucose or lactose, for exmple) of interest (for examples, Yoshioka, et al. U.S. Pat. No. 5,651,869; Fennouh, et al., Increased Paraoxon Detection with Solvents Using Acetylcholinesterase Inactivation Measured with a Choline Oxidase Biosensor, Biosensors & Bioelectronics, 1997, 97-104). The vast majority of U.S. patents issued during the past few years for enzyme biosensors are for designs that employ "ampometric" detection of analyte via transfer of electrons from enzyme to electrode. PA1 (4) Other enzyme biosensor devices that do not rely on electron transfer from enzyme active site to electrode still depend entirely on chemical means for determining the presence of analyte. Color (Gardiol, et al. Development of a Gas-Phase Oxygen Biosensor Using a Blue Copper-Containing Oxidase, Enzyme Microb. Technol. 1996, 347-352) or fluourescence changes (Tolosa, et al. Optical Assay for Glucose Based on Luminescence Decay Time of the Long Wavelength Dye Cy5(T.sub.M) Sensors & Actuators B--Chemical, 1997, 93-99) and pH changes (Blackburn, et al. Potentiometric Biosensor Employing Catalytic Antibodies as the Molecular Recognition Element, Anal. Chem. 1990, 2211-2216) are routinely used as the methods of detection in catalytic biosensor systems. Such requirements for observable chemistries significantly reduce the number of enzymes (and thus analytes) that can be employed in the currently available enzyme biosensor systems. The instant invention does not specifically monitor the enzyme reaction chemistries; rather, the voltage generated by the physical motion of the charged enzyme molecules during interaction with substrate(s) signals that the molecule of interest is present and at what concentration. While one U.S. patent does claim biological sensing as a function of molecular motion (U.S. Pat. No. 5,620,854 to Holzrichter and Siekhaus), this invention requires the use of a scanning probe microscope for determination of macromolecule action. The instant invention requires no such device and relies on the measurement of generated voltage or current.
The biosensor design described in this application is versatile, simple to use, inexpensive to produce, and demonstrates the long-term stability necessary for commercial application. Because the biosensor uses enzymes as the recognition agent, it is able to detect targeted analytes at a speed sufficient for any use. The motion of highly-charged enzyme molecules (as first described by Radmacher, et al. Direct Observation of Enzyme Activity with the Atomic Force Microscope, Science, 1994, 1577-1579) during contact with analyte instantly registers the presence of analyte. Since sensing depends on physical properties shared by all enzymes--and not on any particular enzyme/analyte chemistries--enzymes that perform reactions such as hydrolyses and isomerizations (and thus cannot be detected directly by the current chemistry-dependent biosensing systems) can now be utilized. This change in detection methodology opens the door to the sensing of thousands of new analytes. The biosensor has excellent sensitivity in detection and, in short, has all the properties and production features necessary for use in numerous domestic, military, law-enforcement, medical, and industrial applications.